Bactericidal factors in antisera against various chemotype strains of Salmonella were assayed by means of the spot method. Certain smooth and rough strains were resistant to the killing effect of immune mouse sera when the activity was assayed in an isotonic salt medium and guinea pig complement was used. However, the sensitivity was found to be increased in various degrees by assaying it in a medium containing hypotonic concentrations of NaCl or tris(hydroxymethyl)-aminomethane-HCl (Tris-HCl). Keeping the resistant bacteria in hypotonic solutions before or after treatment with complement increased the titer of antiserum, indicating that the hypotonic solution increases the sensitivity of bacterial cells to the antiserum and/or complement. The optimal salt concentration for the assay of serum-sensitive and -resistant smooth strains was 0.02 M NaCl. With Ra through Rd chemotype strains, 0.1 M NaCl before complement treatment and 0.05 M NaCl after the treatment was the best. Isotonic medium was necessary for the titration of the Re chemotype strain. Specificity of the killing effect which was assayed by the hypotonic spot method was shown by adsorption studies on the immune sera. Use of C4-deficient guinea pig complement resulted in low titers against certain strains of bacteria and high titers were restored by the addition of the C4 component of complement. These results indicate that serologically specific bactericidal factors including antibody can be assayed by the spot method using hypotonic NaCl or Tris-HCl.